Site-Specific Capsid Modification Enables FRα-Directed Retargeting of AAV for Precision Gene Delivery

The clinical translation of adeno-associated virus (AAV)-based gene therapies is often hindered by nonselective tissue transduction, off-target uptake by nontarget cells, and unintended toxicity to healthy tissues. To overcome these challenges, we previously developed a site-specific AAV capsid engineering strategy involving the incorporation of an azide-bearing unnatural amino acid (NAEK) into defined capsid positions, enabling precise, bioorthogonal conjugation of targeting ligands. In this study, we applied this approach to generate a series of folate receptor α (FRα)-targeted AAV2 vectors through covalent tethering of folic acid (FA) at specific capsid residues. FA conjugation at residues S264 + 1 and Q325 significantly enhanced FRα-mediated transduction, yielding a 3-5-fold increase in gene transfer efficiency in FRα-positive tumor cells. Structure-activity relationship analysis revealed that transduction selectivity is governed not only by ligand-receptor binding affinity but also by the spatial location of the conjugation site, which influences competition with the native AAV receptor (AAVR). Importantly, this modular conjugation platform allows for facile replacement of ligands, enabling the rational design of receptor-directed AAV vectors for targeted and cell-specific gene therapy. These findings provide mechanistic insights into capsid-receptor interactions and establish a flexible strategy for precision engineering of AAV-based delivery systems.